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Binding of the flavonoid to MtbHadAB can physically occlude binding of the fatty acid into the substrate binding channel (Fig. 6A).
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Electron density for all the ligands was clear and permitted unambiguous interpretation of the mode of binding of the flavonoids (Fig. 5C).
We tested the binding of these flavonoids to the MtbHadAB complex using an isothermal titration calorimetry (ITC) based assay.
Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA.
The precise binding sites of the flavonoids remain to be determined.
Mapping of the location of this mutation on the structure of MtbHadAB reveals that the mutation is located at the N-terminal end of αHD and lies at the bottom of the flavonoid binding pocket (Fig. 6C).
C1 and Pl proteins containing Myb DNA binding domains regulate downstream genes of the flavonoid synthesis pathway; while B and R proteins containing a basic helix-loop-helix (bHLH) domain are transcriptional activators (Chandler et al. 1989).
Luteolin binds to EAG1 with an EC50 of 8 µM and a Hill coefficient of 4, reflecting the binding of multiple flavonoids.
Therefore, the flavonoids can scuttle binding of the fatty acid or prevent it from binding in a catalytically competent orientation.
The apparent binding affinity of the two flavonoids determined with electrophysiology is higher than the lower limit for the apparent binding affinity determined based on the changes in the fluorescence.
Combinatorial interactions between the transcription factors binding to MYB elements and those binding to other closely located cis-elements are crucial for transcriptional regulation of the flavonoid branch pathway genes [ 31- 33].
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com